There were 3 AF recurrences during the blanking period (90 W 4 s) vs. 1 (50 W). Within each population, one patient was scheduled for a redo-PVI-procedure. The AFEQT score was in favor of the 90 Watt 4 s approach . Conclusion: Within our relatively small studied population, we found hints that in addition to shortening ablation times and radiation exposure without significantly increasing the rate of relevant intraprocedural complications, very high power short-duration ablation (90 W 4 s) provides comparable efficacy rates after one year.Background: Circumferential pulmonary vein isolation (PVI) using radiofrequency ablation (RFA) is a standard of care intervention for patients with symptomatic atrial fibrillation (AF). During follow-up, a substantial number of patients need a redo procedure due to reconnections on the basis of insufficient non-transmural ablation lesions. High-power short-duration ablation (HPSD) is expected to create efficient lesions while causing fewer complications than in conventional RFA settings. The aim of this study was to compare one-year outcome data of very HPSD  to a strategy using 50 Watt HPSD ablation guided by the CLOSE protocol using the Ablation Index (AI), an arbitrary unit composed of power, contact force and ablation time. Methods: We retrospectively analyzed short and long-term (median follow-up 23.2 ± 9.9 months) outcome data from 52 patients that were scheduled for first-do-symptomatic PVI. A very HPSD ablation protocol with 90 Watt and a 4 s duration cut-off was compared to an HPSD CLOSE approach  in terms of freedom from AF recurrence in a long-term electrocardiogram (ECG) over a five days surveillance period. To gain an impression of the subjective sense of wellbeing, the Atrial Fibrillation Effects on QualiTy-of-Life (AFEQT) score was recorded. Results: Overall freedom from AF was found in 81% (90 W 4 s) vs. 87.5% (50 W), ( Atrial fibrillation (AF) is a clinical arrhythmia with a potentially malignant course. In addition to a 1.5–3.5-fold increase in mortality, AF is strongly associated with a significant increase in stroke, congestive heart failure as well as a decrease in quality of life. Furthermore, 10–40% of all AF patients are hospitalized due to their disease at least once per year. Taking that into account, the currently high, and in the future still increasing, AF-prevalence and the resulting individual, health–social and health–economic consequences outline the importance of an optimal therapy strategy . InterveTherefore, our study aims to investigate the efficacy and clinical outcome after one year of very HPSD 90 W 4 s ablation in a high-volume single-center experience.This study is a retrospective two-arm non-randomized observational trial comparing the one-year outcome of patients undergoing PVI using an HPSD RF ablation setting. The study has been performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments. All patients provided written informed consent for the procedures.We included consecutive patients with symptomatic drug-refractory paroxysmal or persistent AF scheduled for RF-based HPSD AF ablation from July 2020 until December 2020. In addition, the 9 patients from the QDOT-FAST trial who were treated at our institution in May 2018 were included in the analysis . AF was All patients underwent either contrast-enhanced cardiac computed tomography with cardiac segmentation or transesophageal echocardiography to exclude intracardiac thrombi prior to ablation. An uninterrupted anticoagulation scheme with direct anticoagulants was used, while for patients on vitamin-K antagonists, the target international normalized ratio was between 2–3. During the procedure, unfractionated heparin was continuously administered to achieve an activated clotting time above 300 s. All procedures were performed under continuous sedation using midazolam, disoprivan and fentanyl. After 3-time venous access, a steerable diagnostic catheter was placed into the coronary sinus. In all patients, a single transseptal puncture with double access to the LA using a steerable 11.7-French sheath was performed . Mapping was performed using a three-dimensional (3-D) electroanatomic mapping system  in combination with a 20-electrode circular mapping catheter  or in selected patients with a multipolar catheter . For PVI, a QDOT MICROTM catheter  in conjunction with a nGEN generator  was used. According to the CLOSE protocol, an interlesion distance of 6 mm was aimed for both groups [Procedural efficacy was evaluated using electrocardiogram patch Holter devices  over a five-day surveillance period which were sent to the patients by mail at least one year after the AF ablation procedure in combination with the AFEQT questionnaire. AF recurrence was defined as a heart rhythm with no discernible repeating P waves and irregular RR intervals (when atrioventricular conduction is not impaired) of equal or more than 30 s in the patch Holter-ECG tracing. In case of symptoms potentially related to a potential AF recurrence episode, an urgent in-house follow-up, including an ECG recording, was ordered. If the standard 12-lead ECG recording showed AF, this was counted as AF recurrence.The Atrial Fibrillation Effect on Quality of Life (AFEQT) questionnaire was administered to all patients during the 1-year follow-up by mail. The AFEQT is a 20-item questionnaire that quantifies four domains of AF-related QoL, including symptoms, daily activities, treatment concerns, and treatment satisfaction, using 7–10-point Likert response scales . An over2 test or the Fisher’s exact test, as appropriate. To evaluate the prognostic value of predefined variables on the study endpoints, Kaplan–Meier curves were calculated with log-rank testing for statistical significance. The result of a statistical test was considered significant for p < 0.05; p values ≤ 0.1 were defined as a statistical trend. SPSS  was used for statistics.Quantitative data are presented as mean ± standard error of the mean (SEM) and were compared using the Student’s t-test for normally distributed data. Qualitative data are presented as absolute and relative frequencies and compared using the Chi2) were ablated using the 90 Watt 4 s very HPSD protocol. In comparison, 21 patients  were treated with the 50 Watt HPSD-CLOSE approach. Between both groups no statistically significant differences within the baseline characteristics could be found . The mean left atrial diameter derived from the transthoracic echocardiographic parasternal long-axis was 38 ± 6 mm in the very HPSD group and 40 ± 6 mm in the 50 Watt HPSD-CLOSE cohort (p 0.38). Among both groups, no statistically significant differences within the concomitant cardiovascular disease were observed  82 ± 18 mL/min/1.73 m2 vs. 77 ± 13 mL/min/1.73 m2, p = 0.46). Consecutively, the CHA2DS2-Vasc-Score was comparable . The medication at discharge was without any statistically significant differences  underwent a first AF ablation procedure using HPSD protocol at our institution. Out of those, 31 , left atrial dwell times , and fluoroscopy times  showed non-significant trends of time-saving for the very HPSD group with the expected highly significant reduction in ablation time . There was no significant difference concerning the first-pass isolation rate  as the complication rate  compared to the HPSD CLOSE approach group. Additional ablations of the right atrial cavotricuspid isthmus for documented typical atrial flutter were performed in 14 patients .  patients in the very HPSD group withdrew consent vs. three out of twenty-one (14.3%) in the 50 Watt HPSD group, p. 0.60. The median follow-up interval was 23.2 ± 9.9 months  without a statistical difference between both groups (p = 0.46). During the 3 months blanking period, 8% of all patients suffered from AF recurrence . On top of those events, one patient from each group suffered from an AF recurrence  and was scheduled for a redo-procedure in the meantime. At the end of the follow-up interval, freedom from atrial fibrillation was similar in both ablation arms   .p = 0.37) with a tendency towards fewer patients with an impaired HR-QoL (AFEQT-OS score < 80) . Unexpected and in contradiction to the findings in the Holter-ECG recordings, only around half of our patients, regardless of the ablation protocol, stated subjective freedom from atrial fibrillation . After adjusting the AFEQT-OS score within the subgroup of patients who reported the subjective impression of freedom from AF, no difference between both groups could be found . The same was true when comparing only the subgroups with the subjective impression of an AF recurrence . Only the patients who mentioned a subjective freedom state from atrial fibrillation independent from their ablation protocol to the group of patients with the subjective impression of an AF recurrence led to a highly significant difference in the AFEQT-OS score . Risk factors were well controlled within both groups and did not differ significantly between both groups at this time . Identically to the baseline, no statistically significant difference in the current medication was seen between both groups .At the end of the follow-up interval, patients in the very HPSD cohort had a slightly higher, though statistically non-significant mean AFEQT-OS score compared to the 50 Watt HPSD CLOSE arm . These later findings are similar but not totally in line with the results of Bortone et al., showing that within their cohort, 90 W 4 s PVI ablation led to similar procedural times due to a lower first pass PVI-rate and higher acute PV reconnection rate . The minp =0.46) and a one-year sinus rhythm maintenance of 82% and 88% (p = 0.52). Those numbers are in line with Bortone’s cohort [p = 0.55). Taking this into consideration, according to the guidelines, the indication for PVI ablation is still based on symptoms and the subjective presence/recurrence of AF goes hand in hand with a reduced quality of life expressed in the statistically lower AFEQT-OS score . Trying to correlate the symptomatic patients to any potentially existing differences in the baseline characteristics as in the risk factor control did not lead to any significant findings. One of the reasons might be that our study cohort was younger and had a CHA2DS2-VASc Score of around 1.5, meaning the patients were less sick than the ones reported in previous studies [Short-term and long-term success rates were comparable between the two groups, with a first-pass isolation rate of 52% and 62% , asymptomatic EDEL may have occurred in the studied population due to the lack of gastrointestinal endoscopic evaluation. Furthermore, the occurrence of asymptomatic cerebral infarction was not assessed in this study.In contemporary practice, very HPSD (90 W 4 s) ablation seems to be safe and effective for achieving freedom from atrial fibrillation recurrences. We found hints that it also shortens ablation times with a tendency towards time-saving within the other measured procedural times if applied in selected patients and experienced hands.


Polyetheretherketone (PEEK) is a thermoplastic polymer that has been recently employed for bone tissue engineering as a result of its biocompatibility and mechanical properties being comparable to human bone. PEEK, however, is a bio-inert material and, when implanted, does not interact with the host tissues, resulting in poor integration. In this work, the surfaces of 3D-printed PEEK disks were functionalized with: (i) an adhesive peptide reproducing [351–359] h-Vitronectin sequence (HVP) and (ii) HVP retro-inverted dimer (D2HVP), that combines the bioactivity of the native sequence (HVP) with the stability toward proteolytic degradation. Both sequences were designed to be anchored to the polymer surface through specific covalent bonds via oxime chemistry. All functionalized PEEK samples were characterized by Water Contact Angle (WCA) measurements, Atomic Force Microscopy (AFM), and X-ray Photoelectron Spectroscopy (XPS) to confirm the peptide enrichment. The biological results showed that both peptides were able to increase cell proliferation at 3 and 21 days. D2HVP functionalized PEEK resulted in an enhanced proliferation across all time points investigated with higher calcium deposition and more elongated cell morphology. Young’s modulus of PEEK is around 3.6 GPa, similar to the value of cortical bone. It is much lower than any metallic material employed in the common bone implant (110–180 GPa) [The increase in life expectancy in Western countries has resulted in a higher incidence of bone and cartilage pathologies. Consequently, the demand for, and optimization of orthopedic implants, has grown . For man180 GPa) . For thi180 GPa) .PEEK Young’s modulus is manageable through additive inclusion: for example, by adding carbon reinforcement in a PEEK matrix, its modulus increases up to 18 GPa, which is superimposable to the values of the cortical bone (18.4–20.7 GPa) . In the The growth of new bone tissue in direct apposition with the implant is desirable for all prostheses but becomes a necessity when the osseointegration process is critical, i.e., for elderly patients or patients suffering from multiple pathologies. With this in mind, modification of the implant surface to enhance the adhesion and proliferation of osteoblasts is a favorable approach. Taking this into consideration, enhancing the bioactivity of PEEK presents a desirable challenge to increase the potential benefit from the wide advantages of this material .To date, a number of physical methods for PEEK surface modification have been proposed, such as thermal spraying, pulsed laser, ion sputtering, sandblasting, and electrochemical methods, which require a lot of optimization work and complex equipment. Our strategy concerns the chemical functionalization of PEEK surfaces with bioactive motifs. Previous studies have used the reduction of the carbonyl groups of PEEK to alcohol groups (PEEK-OH). PEEK-OH is then used to obtain isocyanate groups or amino groups on the surface ,17,18 toIn our grafting strategy, the carbonyl groups already present on the PEEK surface react with an amino-oxy group ad hoc inserted in peptide sequence: the method allows the covalent anchoring of bioactive peptides in a single-step without the use of condensing agents or organic solvents ,20. ThisAcetic acid, acetone, acetonitrile, Fmoc protected Amino acids, Bis-Boc-aminooxy acetic acid (Aoa), Dichloromethane (DCM), N,N-Diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), Ethyl cyano(hydroxyimino)acetate (Oxyma Pure), Methanol, Rink-Amide MBHA resin, Triethylamine (TEA), Triethylsilane (TES), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU) were purchased from Merck Millipore . Diethyl ether, Trifluoroacetin acid (TFA) were purchased from Biosolve . 2-Proponal for HPLC  was purchased from PanReac AppliChem . Ethanol was purchased from Carlo Erba . N-Methyl-2-pyrrolidone (NMP) was purchased from Iris Biotech GmbH .2) was synthesized using the Solid Phase Peptide technique (SPPS) on Tink amide MBHA resin by a Syro I synthesizer . All the amino acids were protected with a Fmoc group on the N-termini and with a Pbf, Trt, Boc, or tBu group on the side chain. Every amino acid was introduced through a double coupling with five equivalents of each amino acid, five equivalents of activating agent HBTU/Oxima Pure, and ten equivalents of DIPEA with respect to the resin reactive groups used for each coupling of 45 min. Aoa was added to the sequence through a double coupling with five equivalents of each amino acid, five equivalents of activating agent HATU/Oxima Pure, and ten equivalents of collidine with respect to the resin reactive groups were used for each coupling of 45 min.Aoa-x-HVP (sequence: H-Aoa-(7-aminoheptanoic acid)-Phe-Arg-His-Arg-Asn-Arg-Lys-Gly-Tyr-NH2O for 1.5 h at room temperature.After the addition of Aoa, the peptide was cleaved from the resin and deprotected from Bis-Boc of Aoa and the side-chain protecting groups using 4.75 mL TFA, 0.125 mL TES, and 0.125 H2O; eluent B: 0.05% TFA in 40% isopropanol and 60% MilliQ water; gradient: from 20 to 50% B in 30 min, flow rate: 0.5 mL/min; detector at 214 nm. tR = 13.215 min).The resin was filtered off, and the solution was concentrated and added with cold diethyl ether. The product was precipitated and filtered. The identity of the peptide was determined by mass spectrometry . The peptide Aoa-x-HVP was purified by RP-HPLC (purity grade 98.74%) and characterized by analytical RP-HPLC Phe indicates a para-Fluoro Phenylalanine residue) was synthesized using the Solid Phase Peptide technique (SPPS) on Tink amide MBHA resin by a Syro I synthesizer . All the amino acids were protected with a Fmoc group on the N-termini and with a Pbf, Trt, Boc, or tBu group on the side chain. Every amino acid was introduced through a double coupling with five equivalents of each amino acid, five equivalents of activating agent HBTU/Oxima Pure, and ten equivalents of DIPEA with respect to the resin reactive groups used for each coupling of 45 min. Aoa was added to the sequence through a double coupling with five equivalents of each amino acid, five equivalents of activating agent HATU/Oxima Pure, and ten equivalents of collidine with respect to the resin reactive groups were used for each coupling of 45 min.Aoa-x-F-HVP (sequence: H-Aoa-(7-aminoheptanoic acid)-(p-F)Phe-Arg-His-Arg-Asn-Arg-Lys-Gly-Tyr-NHAfter the addition of Aoa, the peptide was cleaved from the resin and deprotected from Bis-Boc of Aoa and the side-chain protecting groups as reported for Aoa-x-HVP.2O; eluent B: 0.05% TFA in 40% isopropanol and 60% MilliQ water; gradient: from 20 to 50% B in 30 min, flow rate: 0.5 mL/min; detector at 214 nm. tR = 14.688 min).The resin was filtered off, and the solution was concentrated and added with cold diethyl ether. The product was precipitated and filtered. The identity of the peptide was determined by mass spectrometry . The peptide Aoa-x-F-HVP was purified by RP-HPLC (purity grade 99.36%) and characterized by analytical RP-HPLC  was synthesized using the Solid Phase Peptide technique (SPPS) on Rink amide MBHA resin by a Syro I synthesizer . All the amino acids are of D- type, and they were protected with a Fmoc group on the N-termini and with a Pbf, Trt, Boc, or tBu group on the side chain. Every amino acid was introduced through a single coupling with five equivalents of each amino acid, five equivalents of activating agent HATU/Oxima Pure, and ten equivalents of DIPEA with respect to the resin reactive groups used for each coupling of 45 min. Aoa was added to the sequence through a double coupling with five equivalents of each amino acid, five equivalents of activating agent HATU/Oxima Pure, and ten equivalents of collidine with respect to the resin reactive groups were used for each coupling of 45 min. Aoa-x-D2HVP (sequence: H-Aoa-(7-aminoheptanoic acid)-(DTyr-Gly-DLys-DArg-DAsn-DArg-DHis-DArg-DPhe)After the addition of Aoa, the peptide was cleaved from the resin and deprotected from Bis-Boc of Aoa and the side-chain protecting groups as reported for Aoa-x-HVP.2O; eluent B: 0.05% TFA in 40% isopropanol and 60% MilliQ water; gradient: from 5 to 45% B in 40 min, flow rate: 0.5 mL/min; detector at 214 nm. tR = 32.510 min).The resin was filtered off, and the solution was concentrated and added with cold diethyl ether. The product was precipitated and filtered. The identity of the peptide was determined by mass spectrometry . The peptide was purified by RP-HPLC (purity grade 95%) and characterized by analytical RP-HPLC . The files were then sliced to generate printer-ready G-code and printed using an Apium P155 Peek filament printer . Peek filament  1.75 mm in diameter was extruded through a 0.4 mm diameter nozzle at a temperature of 485 °C onto a print bed with a temperature of 130 °C to create the PEEK disks with a 100% infill at a print speed of 33.3 mm/s. The resulting 3D-printed PEEK disks possess a flat surface and a patterned one.AutoDesk Fusion 360  computer-aided-design (CAD) software was used to design PEEK disks 10 mm in diameter and 4 mm in height and exported in an −5, 10−4, and 10−3 M. After that, the disks were rinsed three times with buffer and three times with MilliQ water.PEEK scaffolds were functionalized with all the peptides. The linkages between the amino-oxy group on the N-terminus of the peptide chain and ketonic groups on the PEEK surface are realized by means of chemoselective ligation. Each peptide was dissolved in 40 mM monobasic sodium phosphate at pH 6 and left to react with PEEK samples for 24 h at room temperature. Peptide concentrations used were 10−8 Torr before introduction in the analysis chamber. The typical vacuum pressure in the analysis chamber during measurements was in the 10−9–10−10 Torr range. The used X-ray radiation is a non-monochromatic Mg Kα(1253.6 eV). The spectra were energy referenced to the C 1s signal of aromatic and aliphatic C atoms having a binding energy BE = 284.7 eV. Curve-fitting analysis of the C1s, N1s, O1s, and F1s spectra was performed using Gaussian profiles as fitting functions after subtraction of a Shirley-type background. Atomic ratio values were calculated from peak intensities. The surface atomic concentrations were determined from photoelectron peak areas using the atomic sensitivity factors reported by Scofield [XPS analysis was performed with a homemade instrument, consisting of preparation and analysis of UHV chambers separated by a gate valve. The analysis chamber is equipped with a six-degree-of-freedom manipulator and a 150 mm mean radius hemispherical electron analyzer with a five-lens output system combined with a 16-channel detector giving a total instrument resolution of 1.0 eV as measured at the Ag 3d 5/2 core level. Samples were introduced in the preparation chamber and left outgassing overnight at a base pressure of about 10Scofield . When se−1, tip radius < 10 nm). AFM measurements were made in triplicate. The probe was characterized by measuring the cantilever spring constant by the Sader method [F is the applied force; adF is the adhesion force; Es is Young’s modulus; sν is the Poisson’s ratio for the sample; R is the radius of the spherical indenter; and δ is the elastic indentation depth. Each surface was indented in different areas, and about three hundred curves were acquired. The tip-sample approaching rate was set to 0.3 µms−1 for all force curves. Young’s modulus was calculated by fitting experimental curves with the DMT model in the elastic region. Elastic modulus distribution was assessed with OriginPro 8.5 software.AFM analyses were carried out on different functionalized PEEK disks at room temperature in order to evaluate the effects of the peptides on the mechanical properties of interfaces separately. The Young modulus was calculated from the collected force–distance curves measured with the NTEGRA AFM . Stiff single-crystal silicon cantilevers with a symmetric tip shape were used , k is the spring constant of the cantilever, and ΔL is the deflection distance (nm). The cantilever spring constants were equal to 0.05 N/m for PEEK-HVP and 0.08 N/m for PEEK-D2HVP.For adhesion force measurements, Au-coated silicon nitride tips were used. The spring constants of the cantilevers used were in the range of 0.003−0.13 N/m. Individual spring constants were calibrated using the Sader method. The adhesion force is taken as the pull-off force required to separate the AFM tip from the surface. The surfaces were probed in different areas, and about three hundred approach-retract cycles were acquired for each system. The tip-sample approaching velocity was set to 0.3 μsThe surface wettability was traced by measuring the static water contact angle (WCA). An OCA30 instrument (Dataphysics) was used at 25 °C and 65% relative humidity. Probe liquid drops of 2 µL of volume were dispended on different zones of each sample surface, and by digital image analysis, the static contact angle, as the tangent to the drop at the point of contact with the surface, both on the right and on the left side, has been acquired. At least three measurements were made for each sample and then averaged. WCA analyses were performed on flat PEEK surfaces.All biological studies were performed on the patterned surface of the 3D-printed PEEK disks.5 cells per disc. Seeded samples were incubated at 37 °C and 5% CO2 for the time duration required or all tests performed. For gene expression evaluation, a density of 2 × 105 HOB cells/per disc was used. Culture media was changed every three days for optimal growth conditions.Human osteoblasts (HOB) were used to evaluate the functionalized PEEK disks. Prior to seeding, PEEK specimens were sterilized in 70% ethanol for 24 h and then washed with PBS. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10 mL of 1 M HEPES, 5 mL of 200 mM L-glutamine, 5 mL of 100× penicillin/streptomycin, 50 mL of Fetal Calf Serum (FCS), 5 mL of minimal essential medium and 0.075 g of solid ascorbate acid. HOB cells were microseeded onto the surfaces of the disks at a density of 1 × 10TM Viability/Cytotoxicity Kit  after 48 h of cell culture. Two PBS washings were performed prior to exposure to live/dead stains. Images were captured with an Olympus IX51 fluorescent microscope. ImageJ software was used to display and merge images of living cells (green fluorescence at 530 nm) and dead cells (red fluorescence at 600 nm) of the same location into one image.Cell viability was assessed using a LIVE/DEADThe morphology of HOB cells on the 3D-printed PEEK structures was observed using SEM  at a voltage of 10kV. Forty-eight hours after seeding, samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for at least 24 h at 4 °C to preserve the structure of cells. Following this, the glutaraldehyde was removed, and the samples were rinsed with 0.1 M sodium cacodylate buffer solution before treatment with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h. The osmium tetroxide was then removed, and samples were further rinsed with 0.1 M sodium cacodylate buffer. 1% tannic acid in 0.1 M sodium cacodylate buffer was used to treat the samples for 1 h. Following this, the samples were dehydrated in a series of aqueous ethanol solutions . For each concentration, the samples were treated twice, and each treatment was 5 min. After dehydration, the samples were air-dried and sputter coated with gold prior to observation under SEM.Calcium deposition was evaluated by Alizarin Red S staining on HOB cells. Alizarin red S is an anthraquinone dye that reacts with calcium and forms an Alizarin Red S/calcium complex that is birefringent. HOB cells were cultured for 21 days, and the disks were then washed in PBS, fixed with 10% formal saline for 15 min, and washed with deionized water three times. 2% Alizarin red S solution was added to the specimens for 15 min, followed by five washes in 50% ethanol. For qualitative assessment, images of the stained scaffolds were taken. A quantitative assessment was conducted using Lee et al. protocol . The bouTM kit. For each sample, three replicates were measured with a microplate reader , which measured the fluorescence values at 570 nm test wavelength and 630 nm reference wavelength.The proliferation of osteoblasts cultured on functionalized and non-functionalized PEEK disks was assessed at 1, 4, 7, 14, or 21 days using the AlamarBlueSpecific mRNA transcript levels coding human SRY-Box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), Alkaline Phosphatase (Alp), and Osteocalcin (Oct) were quantified in osteoblast cells cultured for 24 h on functionalized and non-functionalized PEEK. At the end of incubation, the total RNA was extracted using the SV Total RNA Isolation System kit . Contaminating DNA was removed by DNase I digestion . cDNA synthesis and subsequent polymerization were performed in one step using the iTaq Universal SYBR Green One-Step Kit . The reaction mixture contained a 200 nM forward primer, 200 nM reverse primer, iTaq universal SyBR Green reaction mix, iScript reverse transcriptase, and a 200 ng total RNA. Real-time PCR was performed using the ABI PRISM 7700 Sequence Detection System . Data were quantified by the ∆∆CT method using human Gapdh as the reference gene. Target and reference genes were amplified with efficiencies near 100%. The oligonucleotides used for PCR are listed in All assays were performed at least in triplicate, and all values are reported as mean ± standard deviation.TM and gene expression results were compared with two-way ANOVA tests, followed by Tukey’s post-hoc test.Data were analyzed and processed with Prism (GraphPad Software 9). Alizarin Red S results were compared by using a one-way analysis of variance (ANOVA) test. Tukey’s multiple comparisons test was carried out to confront every condition. AlamarBlueThe significance level applied was 5%.PEEK disks were functionalized with Aoa-x-F-HVP to allow direct individuation of the surface peptide through XPS fluorine atom detection.−5 M to 10−3 M), the surface peptide presence increases  are reported in C1s spectra a are madN1s spectra b are madO1s spectra c are madPeptide immobilization on the PEEK surface is evidenced by the appearance of the N1s signals typical of the peptide; the amount of surface peptide is similar in the two samples.As shown in i.e., the slight decrease of the static water contact angle, Both peptides on PEEK disks slightly increase the seeming hydrophilicity of the polymer surface  Phe. The presence of fluorine as a marker allowed for easier XPS analysis. From the XPS data, it can be deduced that the increase in the concentration of peptide in solution leads to an increased surface peptide density, as evidenced by the enhancement of the F/C ratio when passing from the operating concentration of 10The advantage of the anchoring method proposed via oxime is that it is extremely simple and absolutely free of unwanted reactions or side effects (persistence of condensing agents or traces of organic solvents). We had evidence of the chemistry solidity of this anchoring strategy in chemoselective conjugations where the conjugate identity determination was possible by MALDI-TOF spectrometry . FurtherThe enrichment of the surface with adhesive peptides was ascertained with XPS analysis. From the N/C ratios, it is clear how the two peptides reacted differently: considering that D2HVP has a double sequence then HVP (double nitrogen content), it can be estimated that the surface concentration of D2HVP is about 1/3 compared to that obtained using the peptide HVP.The presence of peptides modified the surface properties of the material, making PEEK more hydrophilic and, therefore, more suitable for interaction with cells. The evaluation of Young’s modulus by AFM showed that functionalization of the surface increases its stiffness. The result is particularly relevant following the functionalization with the shorter HVP peptide. The hypothesis is that this smaller peptide compared to D2HVP, is able to fill the gaps between the surface polymer chains while its retro-inverted dimer does so only in part due to the greater steric hindrance. Both peptides increased the adhesion forces with the AFM cantilever compared to the control. In particular, the presence of the HVP peptide doubles the adhesive force with the cantilever tip compared to PEEK alone, while the D2HVP peptide tripled the adhesion force compared to the control. This may be due to the longer sequence of the retro-inverted dimer compared to HVP.The Live and Dead staining at 48 h showed a large number of viable cells attached to all the surfaces, and only a few red dead cells were visible on the HVP sample but were absent on the D2HVP sample. The elongated morphology, index of spreading, and, therefore, adhesion were observed on the control PEEK, and the PEEK was functionalized with D2HVP. Cells on PEEK-HVP appeared more rounded, indicating more metabolically active cells.TM test. On day one, an increase in the number of adhered cells was observed on both functionalized surfaces when compared to the control by about 300% in accordance with the adhesive nature of the sequences [Quantification of the cells at different time points was assessed with the AlamarBlueequences . At the equences .The calcium ion deposition was greater on the D2HVP peptide, indicating an increase in mineralization with respect to its monomer and control: the difference, however, was not significant. Furthermore, HVP and D2HVP functionalization significantly enhanced the gene expression of Sox9 at one day from cell seeding, and although it induced a down-regulation of Runx2 at one day, this is inverted at days 7 and 14, suggesting a positive trend over time (for HVP only). Expression of Sox9 has been observed in in vitro osteogenic differentiation of human Mesenchymal Stem Cells (hMSCs) . HoweverScaffolds play a significant role in cell adherence, a major contributing factor to subsequent morphology, proliferation, and maturation of cells. The presence of bioactive sequences on the PEEK surface allowed the design of an engineered surface able to promote the proliferation and differentiation of human osteoblasts. Results have shown an increase in adherence, proliferation, and maturation of osteoblasts. To further examine and elucidate the mechanistic actions of these peptides, future studies will explore their role in the induction of stem cells towards the osteogenic lineage and examine regulatory signaling pathways in vitro.


There are many different chronic lymphoblastic leukemia (CLL) survival prediction models and scores. But none provide information on expression of immune-related genes in the CLL cells.We interrogated data from the Gene Expression Omnibus database  and International Cancer Genome Consortium database  to develop an immune risk score (IRS) using Least absolute shrinkage and selection operator (LASSO) Cox regression analyses based on expression of immune-related genes in CLL cells. The accuracy of the predicted nomogram we developed using the IRS, Binet stage, and del(17p) cytogenetic data was subsequently assessed using calibration curves.p < 0.001) and 98%  vs. 92%  in two datasets. The IRS was an independent survival predictor of both datasets. A calibration curve showed good performance of the nomogram. In vitro, the high expression of CDKN2A and SREBF2 in the bone marrow of patients with CLL was verified by immunohistochemistry analysis (IHC), which were associated with poor prognosis and may play an important role in the complex bone marrow immune environment.A survival model based on expression of 5 immune-related genes was constructed. Areas under the curve (AUC) for 1-year survivals were 0.90  and 0.75  in the training and validation datasets, respectively. 5-year survivals of low- and high-risk subjects were 89%  vs. 6% (0, 17%; The IRS is an accurate independent survival predictor with a high C-statistic. A combined nomogram had good survival prediction accuracy in calibration curves. These data demonstrate the potential impact of immune related genes on survival in CLL. IGHV) mutation state, cytogenetic abnormalities, especially del(11) and del(17p), serum β2-microglobulin, lymphocyte doubling time and others. These models and scores have only modest accuracy with C-statistics of 0.60–0.77  including Binet and Rai staging systems, MD Anderson Cancer Center 2007 index score, CLL International Prognostic Index (CLL-IPI), Barcelona-Brno score, International Prognostic Score-A (IPS-A), CLL-01 and a tailored approach . These mWe developed a survival predictive model we termed the Immune Risk Score (IRS) by analyzing immune-related gene expression data in CLL cells from two publicly available datasets. We evaluated the model in multivariable analyses and verified it in a validation dataset. We combined the IRS with Binet stage and del(17p) cytogenetics data to develop a survival prediction nomogram.1 for extracting all immune genes in the GEO dataset and the ICGC dataset, to obtain a list of candidate immune genes for further study. The workflow is summarized in Data from 2 CLL datasets including 642 subjects with micro-array survival data, GSE22762 and CLLE-ES , were obp < 0.05. We used Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analyses to identify the best weighting coefficient of immune-related genes for survival. The maximum likelihood estimate of penalty after 1,000-fold cross-validation was determined and the 1-SE criterion was used to determine the optimal value of the penalty parameter λ. These data were used to develop the IRS. The CLLE-ES dataset was used as validation set.The GSE22762 dataset was used for training to construct the IRS model. Univariable Cox regression analyses were used to identify immune-related genes significantly correlated with survival at Prediction accuracy of the IRS was quantified using time-dependent receiver-operating characteristic (ROC) curves and the area under the curve (AUC) method. Subjects were divided into high- and low-risk cohorts by the optimal cut-off of risk score in each dataset. Survivals of subjects in the high- and low-risk cohorts were compared by Kaplan–Meier curves with log-rank tests. The GSE22762 and CLLE-ES datasets had clinical co-variates which were included in uni and multivariable Cox regression analyses.We developed a survival prediction nomogram by combining the IRS with Binet stage and del(17p) cytogenetics data. In the nomogram, each co-variate is assigned a point and added to give a total point corresponding to 1-, 3-, or 5-year survival. The calibration curve showed the relationship between the predicted value and the measured value of the nomogram in the training dataset. The IRS, clinical and laboratory co-variates and nomogram were compared with the time-dependent receiver operating characteristic curve (time-ROC curve) for 5-year survival.We collected 44 newly diagnosed CLL patients, whose diagnosis was confirmed by histopathology following bone marrow aspiration procedures taking place between 2011 and 2020 at Sun Yat-sen University Cancer Center (SYSUCC), Guangzhou, China. The other inclusion criteria were: No acute infection 2 weeks prior to enrollment; no chronic inflammation or autoimmune disease; and no consumption of immunosuppressive or immunomodulatory drugs. All patients were followed-up until September 2020 through hospital records or phone contact with the patients or relatives who were aware of their illness. This study was approved by the Research Ethics Committee of SYSUCC (Approved number B2021-044-01) and was conducted in accordance with the Declaration of Helsinki.+ T cells, CD8+T cells, and neutrophils were selected to further explore the relationship between immune genes and the bone marrow immune environment.We verified two immune-related genes (CDKN2A and SREBF2) with the larger weight coefficient according to IRS model, by immunohistochemistry analysis. Several types of immune cells, including monocytes, CD4+ T cells , CD8+ T cells , monocytes , neutrophils , and two immune-related genes: the sterol regulatory element binding transcription factor , and the cyclin dependent kinase inhibitor 2A . Every five duplicate slide was analyzed by two independent researchers in a blinded manner. For the immune cells, researchers recorded the number of positive immune cells and calculated the density of immune cells using Image J software . For the immune-related genes, we recorded the staining area and degree of immune protein expression using a 1–3-point system. The points were added up to obtain a total immunohistochemistry analysis score for each immune-related gene.Briefly, the paraffin-embedded bone marrow puncture specimens were stained using antibodies against CD4P < 0.05 were used in subsequent multivariable Cox regression analyses. SPSS software version 24.0 , R software version 3.6.2 , and Perl version 5.24.3  were used for statistical analyses. A two-sided P < 0.05 was considered significant.Subjects were divided into high- or low-risk cohorts at the optimal cut-off IRS which was determined by ROC curves analyses. Prediction accuracy was quantified using time-dependent ROCs and AUCs calculated to determine the Concordance C-Statistic. Survival was defined as the interval from diagnosis to death from any cause. Time-to-therapy (TTT) was defined as the interval from diagnosis to start of 1st or next treatment. A significant proportion of patients in GSE22762 were not analyzed at diagnosis but at a more advanced disease stage and in relapse. Survival and TTT in those patients were calculated as the interval from measurement to the respective event. Survivals were compared in Kaplan–Meier curves using the log-rank test. In univariable Cox regression analyses co-variates with CDKN2A, SREBF2, NRIP1, BCL11B, and SIRT1  + (0.27 × SREBF2 RNA expression) − (0.04 × SIRT1 RNA expression) − (0.11 × BCL11B RNA expression) − (0.26 × NRIP1 RNA expression).IRS = . Comparable survivals in the validation dataset were 98%  vs. 92% . The IRS in each dataset was an independent prognostic factor in multivariate Cox regression analysis , 0.78, 0.99), 0.84 , and 0.88  data shown in . There . The nomP < 0.05; + T cells, CD4+ T cells, CD14+ monocytes, and CD66b+ neutrophils, whereas CDKN2A expression negatively correlated with the presence of CD8+ T cells, CD4+ T cells, and CD66b+ neutrophils , promotes the anti-tumor response of T- and NK-cells and prevents immune evasion of colon cancer cells  but down-regulated in breast and liver cancers  stem cells by enabling over-acetylation and reducing the activity of TET2  in the bone marrow immune environment correlated positively with good CLL prognosis  limiting our multivariable analyses. 2nd, survival of subjects in the validation set was significantly better than in the training dataset reflecting different therapies and disease stage, which has the advantage of allowing us to term the IRS a prognostic rather than predictive score. However, the very good survival of the high- and low-risk cohorts in the validation cohort decreased but did not negate discrimination power of the IRS. 3rd, the large differences between the datasets make it impossible to define a uniform cut-off. Future studies are necessary to establish an optimal cut off.In conclusion, we developed and validated a prognostic survival model for CLL based on the expression of 5 immune-related genes which had high accuracy. We also developed a survival nomogram by adding Binet stage and del(17p) cytogenetics data to the IRS which improved the C-statistic. Two immune-related genes were predicted and validated using immunohistochemistry analysis experiments, which may play an important role in the complex bone marrow immune environment.The original contributions presented in this study are included in the article/This study protocol was reviewed and approved by the Data Access Committee  from the ICGC database, without written informed consent. This study was also approved by the Research Ethics Committee of SYSUCC . Research was conducted in accordance with guidelines for human research and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.H-YH wrote the original manuscript. YL, RG, H-YH, and YW reviewed and edited the manuscript. H-YH and YW performed the software analyses. TH, J-ZW, and LL collected the data. YL and H-XL were responsible for the research design, project administration, and funding. All authors reviewed the final manuscript and agreed to submit for publication.


TPM in Haliotis discus hannai (Hdh-TPM) was 2160 bp, encoding 284 amino acids, and contained a TPM signature motif and a TPM domain. Gene ontology (GO) analysis based on the amino acid sequence predicted Hdh-TPM to have an actin-binding function in the cytoskeleton. The 3D analysis predicted the Hdh-TPM to have a coiled-coil α-helical structure. Phylogenetically, Hdh-TPM formed a cluster with other TPM/TPM1 proteins during analysis. The tissue-specific mRNA expression analysis found the higher expression of Hdh-TPM in the heart and muscles; however, during embryonic and larval development (ELD), the higher expression was found in the trochophore larvae and veliger larvae. Hdh-TPM expression was upregulated in fast-growing abalone. Increasing thermal stress over a long period decreased Hdh-TPM expression. Long-term starvation (>1 week) reduced the mRNA expression of Hdh-TPM in muscle; however, the mRNA expression of Hdh-TPM was significantly higher in the mantle, which may indicate overexpression. This study is the first comprehensive study to characterize the Hdh-TPM gene in Pacific abalone and to report the expression of Hdh-TPM in different organs, and during ELD, different growth patterns, thermal stress, seasonal changes, and starvation.Tropomyosin (TPM) is a contractile protein responsible for muscle contraction through its actin-binding activity. The complete sequence of  Myosin attaches to the actin filament and performs a power stroke, which results in the shortening of the sarcomere. During relaxation, as Ca2+ levels drop, this process is reversed [2+ concentrations, troponin locks TPM in position on the F-actin, where it obstructs the myosin-binding site, thus preventing the contraction of the sarcomere [Tropomyosin (TPM), a dimeric-helical coiled-coil protein , is thouechanism . During reversed . At low arcomere . During arcomere . Muscle arcomere . Contracarcomere . Muscle arcomere .In invertebrates, TPM has been reported to occur during embryonic and larval development (ELD), indicating that TPM may play an important role in ELD and adult life . HoweverThe temperature dependence of physiological processes in marine ectotherms is well recognized . The heaH. discus hannai, is extensively cultured among the available abalone species due to its high value and commercial importance not only in South Korea but in many Asian countries [Hdh-TPM was isolated and characterized from Pacific abalone muscle tissue, and the changes in mRNA expression were documented in different developmental stages, growth modes, and experimental conditions.Abalones are a group of marine gastropods that belong to the Haliotidae family. Approximately 56 abalone species are found in the temperate and tropical coastal habitats of the United States, Australia, and East Asia ,20; howeountries ,24,25,26ountries , the groountries . In thisThree-year-old sexually mature male and female Pacific abalones with a mean body weight of 120.4 ± 0.61 g and a mean shell length of 84.06 ± 0.32 mm were collected from sea cages from Jindo-gun, South Korea. The collected Pacific abalones were transported using an icebox to the molecular physiology laboratory at the Department of Fisheries Science, Chonnam National University, Gwangju, South Korea.2. For cloning, a total of 12 Pacific abalones were sacrificed, and their hemocyte, cerebral ganglion, pleuropedal ganglion, testis, ovary, mantle, muscle, gill, heart, and digestive gland tissues were collected. The collected tissue samples were washed with phosphate-buffered saline , immediately snap-frozen in liquid nitrogen, and stored at −80 °C until total RNA extraction.Prior to tissue sample collection, abalones were subjected to anesthetization using 5% MgClArtificial fertilization of Pacific abalones was performed following the procedure described by Hanif et al. . BrieflyHdh-TPM in Pacific abalones, a heat treatment was performed. Adult Pacific abalones  were purchased from an abalone farm in Jindo-gun, South Korea and were transported to the abalone hatchery in Yeosu, South Korea. The abalones were reared in culture tanks with a maintained water temperature of 20.4 ± 0.3 °C for two weeks to allow recovery from the stress of transportation. The abalones were then divided into three groups and kept for a 48 h acclimation period in 15 °C, 25 °C, and 30 °C seawater controlled by a digital temperature controller. No food was provided during this heat-stress treatment. Tissue samples from the muscle and mantle were collected after 1, 6, 12, 24, and 48 h and were washed with 0.1 M PBS. After washing, the tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C until total RNA extraction. Initial control samples were also collected and stored following this process.To observe the stress response of 2 and sacrificed for sample collection. Mantle and muscle tissues were collected, washed with 0.1 M PBS, and stored at −80 °C after being snap-frozen in liquid nitrogen.Three-year-old fast-growth and slow-growth Pacific abalones were collected from the culture area. A total of ten abalones of each growth type were anesthetized with 5% MgClThree-year-old Pacific abalone having a body weight of 100.2 ± 0.56 g and a mean shell length of 80.03 ± 0.31 mm were randomly collected from the sea cages in Jindo-gun, South Korea, and transported to the abalone hatchery in Yeosu, South Korea. The collected abalones were reared in rectangular cemented tanks with running seawater flow and adequate feed supply for an adjustment period. After a month, tissue from the mantle and muscle of ten Pacific abalones was collected, following the procedure mentioned above, and stored for total RNA extraction. Additional abalones were reared without feeding, and tissue samples (mantle and muscle) were collected every seven days for three weeks and seven days after refeeding, following the procedure mentioned above, and stored at −80 °C until total RNA extraction.2. Mantle and muscle tissues were collected and washed with 0.1 M PBS. After washing, the tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C until total RNA extraction.Three-year-old Pacific abalones were collected seasonally  and sacrificed after anesthetizing with 5% MgCl® RACE 5′/3′ Kit , 5′- and 3′-RACE cDNA was synthesized from 1 μL of tRNA from muscle tissue. Extraction of total RNA and cDNA synthesis was conducted following the manufacturer’s protocol.Total cellular RNA was extracted from the collected tissue samples using an ISOSPIN Cell and Tissue RNA kit . First stand cDNA was synthesized from 1–4 μL of total RNA using oligo (dT) primers (OdT) (Sigma) and a superscript III First-strand cDNA synthesis kit . Using a SMARTerHdh-TPM, a reverse transcription polymerase chain reaction (RT-PCR) was conducted using cDNA from the muscle tissue, a set of primers (forward and reverse), and GoTaq® DNA Polymerase . The primer set used in this study was designed from the TPM nucleotide sequence from H. discus discus (GenBank Accession no. AB444939.1). All primers used in this experiment are presented in Hdh-TPM RT Fw) and reverse (Hdh-TPM RT Rv) primers (1 μL each), GoTaq reaction buffer (10 μL), dNTP mix (1 μL), DNA polymerase (0.25 μL), and ultrapure water (35.75 μL). The RT-PCR thermal cycling condition was as follows: initial denaturation for 3 min at 95 °C; followed by 35 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 58 °C, and extension for 45 s at 72 °C; and a final extension for 5 min at 72 °C. Obtained PCR products were subjected to 1.2% agarose gel electrophoresis, and positive band was purified using a Wizard® SV Gel and PCR Clean-Up System kit . The purified DNA was then ligated into the pGEM®-T Easy Vector  and transformed into DH5α chemically competent Escherichia coli . The positive clones were selected for plasmid DNA purification using a Hybrid-QTM Plasmid Rapidprep mini kit  and were sequenced at Macrogen .For the partial sequence cloning of Hdh-TPM, a rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) was performed using a SMARTer® RACE 5′/3′ kit . A set of gene-specific 5′- and 3′-RACE primers were prepared from the obtained partial sequence of Hdh-TPM, including a 15 bp (GATTACGCCAAGCTT) overlap at the 5′-end of the primer sequence. The 5′- and 3′-RACE-PCRs were performed using 2.5 μL of 3′- or 5′-RACE cDNA, 1 μL of sense (3′RACE) or antisense (5′RACE) RACE primers, 5 μL of a universal primer mix (UPM), 1 μL of SeqAmp DNA polymerase, 25 μL of SeqAmp buffer, and 15.5 μL of PCR-grade water. Touchdown PCR was carried out with 30 cycles for both 3′-RACE and 5′-RACE. The thermal cycle conditions for the RACE-PCR were maintained according to the conditions prescribed in the kit. The PCR products were subjected to gel electrophoresis (as described previously), and purification was performed using a NucleoSpin® Gel and PCR Clean-up kit . The purified products were ligated into a linearized pRACE vector and transformed into stellar competent cells. Positive clones were purified and sequenced at Macrogen, as previously described, for the partial sequence. Finally, the 5′-RACE sequence, the initially cloned partial cDNA fragment, and the 3′-RACE sequence were combined and trimmed to obtain the full-length sequence.To obtain the full-length sequence of Hdh-TPM were analyzed by using several online tools. Potential protein encoding segments and open reading frames (ORFs) were predicted from the nucleotide sequence using the online tool ORFfinder . The molecular weight of Hdh-TPM protein and the isoelectric point (pI) were computed using the online tool ProtParam . The protein structure and gene ontology of the Hdh-TPM protein were predicted using the online protein structure prediction server Contact-guided Iterative Threading ASSEmbly Refinement (C-I-TASSER) . The functional domains of the Hdh-TPM protein were ascertained using the SMART server  and the Motif scan . The conserved motifs of the Hdh-TPM protein were discovered using the Multiple Em for Motif Elicitation (MEME) online tool . The coiled-coil region in the protein sequence was predicted using the online tool Waggawagga . The multiple sequence alignment was performed using the MEGA version 11.0.13 software and visualized using Jalview version 2.11.1.7.The complete nucleotide and protein sequence of the cloned The amino acid sequence from Hdh-TPM was aligned with the other related protein sequences using the online tool ClustalW. The phylogenetic tree was constructed and edited using the MEGA software (version 11.0.13) with a maximum likelihood algorithm.The predicted three-dimensional (3D) structure of Hdh-TPM was constructed using the previously mentioned protein structure and the functional prediction program, C-I-TASSER. The visualization of the 3D structure was performed using the UCSF ChimeraX software (v. 1.2.5).Hdh-TPM in different tissues of Pacific abalone. A total of ten different tissues from Pacific abalones, including cerebral ganglion, pleuropedal ganglion, gill, testis, ovary, digestive gland, hemocyte, heart, muscle, and mantle, were used to observe the difference in Hdh-TPM expression. Due to the stable expression in different organs, β-actin from H. discus hannai (accession no. AY380809) was used as the internal control. A 20 μL reaction mixture was used, containing 1 μL of the cDNA template, 1 μL each of the forward and reverse primer, 10 μL of the 2× Prime Taq premix , and 7 μL of ultrapure water. The RT-PCR thermal cycling conditions were maintained for the semiquantitative RT-PCR.Semiquantitative RT-PCR was performed using gene-specific forward and reverse primers to observe the expression patterns of the Hdh-TPM, a quantitative real-time PCR (qRT-PCR) analysis was performed using different tissues from Pacific abalones. The expression levels of Hdh-TPM were observed in the different organs of adult Pacific abalone and the various embryonic and larval developmental stages of Pacific abalone. All qRT-PCRs were conducted following the protocol previously described by Hanif et al. [To quantify the relative mRNA expression levels of f et al. . In qRT-p-value less than 0.05. All graphs were prepared using MS Excel and GraphPad Prism 9.3.1 software.The values of mRNA expression were analyzed statistically and expressed as the mean ± standard error. Changes in relative mRNA expression were computed using GraphPad Prism (version 9.3.1) software by a nonparametric ANOVA analysis. Statistical significance was set at a Hdh-TPM was cloned from the muscle tissue of H. discus hannai and designated as Hdh-TPM. The full-length sequence of the Hdh-TPM cDNA (GenBank accession OM937906.1) was 2160 bp long, including a poly-A tail  analysis using the C-I-TASSER server predicted that the Hdh-TPM protein acted on the following biological process: single-organism cellular process (GO: 0044699) with a C-scoreGO of 0.78; the following cellular component: intracellular organelle (cytoskeleton part) (GO: 0043229) with a C-scoreGO of 1.00; and the following molecular function: actin-binding cytoskeletal protein (GO: 0008092) with a C-scoreGO of 0.48 .H. discus discus and H. asinina.An unrooted phylogenetic tree was constructed using the maximum likelihood method based on the amino acid sequences of TPM proteins from various species, and showed four major groups: TPM, TPM2, TPM3, and TPM4 . The HdhHdh-TPM gene was highly expressed in the heart and muscle tissue of Pacific abalone, H. discus hannai. The expression was found to be very weak in gill tissue (Hdh-TPM was significantly higher in the heart and muscle (p < 0.05) than in the pleuropedal ganglion, branchial ganglion, testis, ovary, digestive gland, and mantle tissues (p < 0.05). Supporting data were also found from the semiquantitative RT-PCR expression analysis.In the tissue distribution analysis, the l tissue A. Simila tissues B. SignifHdh-TPM was expressed ubiquitously. Hdh-TPM mRNA was highly expressed in the unfertilized eggs, but the expression level was significantly reduced immediately after fertilization (fertilized eggs). However, there was some increased level of expression found during the cell division phases. The expression level was lowest in the two-cell stage compared with other examined stages. After fertilization, veliger showed the maximum expression followed by the first larval stage (trochophore), blastula, juvenile, four-cell, and fertilized egg stages  stages, g stages .Hdh-TPM were differentially expressed in the fast- and slow-growing abalones. The relative mRNA expression level of Hdh-TPM in the muscle tissue of the fast-growing group showed higher expression compared with the slow-growing group during the study period  compared with controls and the other time points. Exposure to 25 °C caused the highest upregulation of Hdh-TPM, which doubled and tripled the ones induced by, respectively, the 15 °C and 30 °C treatments. After 12 h, the expression of Hdh-TPM at 15 °C was reduced and stable for all the time points up to 48 h. The expression of Hdh-TPM after six hours at 25 and 30 °C reduced gradually for all the subsequent time points tested. The minimum expression was found to be after 48 h of heat stress for all tested temperatures  conditions, significant differences were observed in the expression of arvation . In contTPMs are a large family of actin-binding filament proteins extensively found in vertebrates and invertebrates . TPM firHdh-TPM from the muscle tissue of Pacific abalone had a high aliphatic index, indicating a thermophilic protein. The main features of this protein are the presence of a long α-helical coiled-coil TPM domain and a C-terminus TPM signature. Generally, coiled-coils are involved in a much wider range of biological functions, including signal transduction, regulation of gene expression, oligomerization, and transport of other molecules [Hdh-TPM might be involved in muscle contraction. The coiled-coil signature motif also performs a role in steric regulation [Successfully cloned olecules . Since tgulation  and is cgulation .Hdh-TPM in Pacific abalone. In contrast, a lower expression was found in the non-contractile gill, suggesting that Hdh-TPM has no function regarding contractility in this organ. Some researchers have reported that TPM functions in ovarian contraction [Hdh-TPM was relatively low in the ovary. The probable reason for this lower expression may be related to the ovarian development stage.The tissue-specific mRNA expression analysis showed higher expression in the heart and muscle tissues. These three organs are contractile organs, indicating the contractile function of traction ; howeverHdh-TPM was dynamically expressed throughout the ELD stages of Pacific abalone. Although the present study found that the highest expression was in the unfertilized eggs, the role of Hdh-TPM at this stage remains unclear [Hdh-TPM in trochophore and veliger larvae suggests that it may play a role in complex cellular processes, including muscle development. In the small abalone, Haliotis diversicolor, the encapsulated retractor muscle appears in the trochophore stage and is enlarged in the veliger stage [Hdh-TPM may be involved in these activities because the GO analysis indicated that it was involved in the cellular component organization and development process, and the abalone retractor muscle appears in the trochophore stage. unclear . The higer stage . In abalHdh-TPM was found to be upregulated in the mantle and muscle tissues of fast-growing abalones. However, the expression was significantly higher in the muscle compared with the mantle. Since the tissue was collected from fast-growing and slow-growing abalones of the same age from identical parents and cultured in the same environmental conditions, including food availability, there was less chance of influencing their growth pattern by these factors. Therefore, the growth variability in the abalone muscle was most likely due to the activation of a growth inhibitory gene, myostatin, which is somewhat regulated during muscle contraction via the actin-binding activity of Hdh-TPM. Skeletal muscles are some of the most dynamic tissues involved in voluntary contractions [Hdh-TPM indicated a higher actin-binding activity and a higher muscle contraction rate. The information regarding muscle contraction or the effects of tropomyosin on fast- and slow-growing animals is scarce. This is the preliminary study about the functional activity of tropomyosin in different-growth-type animals.The higher expression of TPM in contractile tissue indicates a higher actin-binding activity, and the lower expression indicates a low actin-binding activity regulating contractility. The expression of ractions . The higHdh-TPM in the muscle and mantle at different ambient temperatures. In the muscle tissue, the comparatively lower Hdh-TPM mRNA expression after one hour of heat stress is indicative of a reduction in actin-binding activity and muscle contraction. Although the underlying cause of the decreased Hdh-TPM mRNA expression after one hour of acclimation to the controlled temperature is unknown, it may be related to sudden heat stress and stress induced by the animal handling during the experimental setup. The optimum muscle contraction for jack mackerel was found to be 18 °C, and the contraction rate was found to be stable at temperatures ranging from 15 to 22 °C [Hdh-TPM at 25 °C after six hours of stress treatment may indicate overexpression. Overexpression of Hdh-TPM at 30 °C may occur earlier than six hours (possibly after 3–4 h of heat stress treatment); it began to decrease after reaching the peak.The temperature dependence of physiological processes in marine ectoderms is well recognized . Hdh-TPMto 22 °C . Additioto 22 °C . The lowto 22 °C  within tHdh-TPM showed differential expression in the mantle and muscle tissues. Seasonal changes are temperature-dependent, with winter temperatures ranging from 3 to 8 °C, spring and autumn temperatures ranging from 13 to 18 °C, and summer temperatures ranging from 23 to 28 °C in Korea [Hdh-TPM was observed, indicating muscle contraction in these seasons.In response to seasonal change, in Korea . GeneralHdh-TPM during the first week of starvation in the mantle and muscle tissues. In crickets, upregulated expression of TPM was also previously reported during short-term starvation [Hdh-TPM significantly increased in the mantle and decreased in the muscle (not significant) until refeeding. The reason for the significantly higher expression in the mantle is unknown; however, the lower expression in the muscle may be related to reduced contraction due to a decreased Ca2+ supply that mainly comes from dietary sources [Hdh-TPM and muscle contraction rate and, thus, negatively affects muscle growth.Starvation is one of the common nutritional stresses that can affect abalone body physiology, by influencing the expression of several genes . The prearvation . However sources . Short-t sources . DecorinHdh-TPM also indicated a contractile function that might play a role in muscle growth. The expression of Hdh-TPM in early development indicates that Hdh-TPM may be involved in ELD, especially in the development of the retractor muscle. In response to thermal stress and seasonal variation, the expression of Hdh-TPM also indicated contractile activity. Reduced expression of Hdh-TPM in Pacific abalone muscle tissue during starvation was also indicative of lower muscle contraction because the Ca2+ available in food required for contraction was not supplied. Altogether, the findings are indicative of a potential function of TPM in muscle contraction and, thus, growth in the abalone Haliotis discus hannai.TPM is a well-known muscle protein that largely controls muscle contraction by binding to actin. Coiled-coil α-helical structures of the Hdh-TPM protein sequence may be involved in this activity. GO and the functional domain of the cloned


The long-term overall survival of children with T-cell acute lymphoblastic leukemia (T-ALL) is limited to approximately 80–85% because of a high incidence of relapse after achieving remission with intensive chemotherapy and hematopoietic stem cell transplantation (HSCT). Novel treatment strategies inducing long-term remission are needed to improve the outcome. Histone deacetylase inhibitors (HDACis) have been reported to be effective in a series of T-ALL cases. Preclinical studies suggested that T-ALL cells are sensitive to Chidamide, which is a selective HDACi.This preliminary clinical study evaluated the efficacy and safety of Chidamide in combination with chemotherapy or post-HSCT for children with T-ALL at a dose of 0.5 mg/kg weight of patient twice per week for at least 6 months.In total, 27 children with a mean age of 7.88 years were included. The high-risk proportion was 66.7%. After a median follow-up period of 37.8 months (9.5–67.9 months), the overall survival and event-free survival in the patients treated with Chidamide were 94.1 and 95.2%, respectively. All patients except two maintained persistent remission with <0.01% blast cells in minimal residual disease.The combination therapy with Chidamide in a case series of T-ALL shows the promising clinical efficacy and good safety in children.https://www.chictr.org.cn/, identifier ChiCTR2000030357. T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 12–15% of all ALL cases in childhood. The long-term overall survival (OS) of children with T-ALL is limited to approximately 80–85% because of a high incidence of relapse after achieving remission with intensive chemotherapy . CurrentStudies have suggested that high expression of Histone deacetylase (HDAC) in childhood T-ALL is associated with poor prognosis, which indicates that HDAC inhibitors (HDACis) may be used to treat T-ALL . HDACis Based on these rationales, for the treatment of children with T-ALL at our institution, Chidamide has been added to the chemotherapy and for post-HSCT relapse prophylaxis. Here, we report the clinical efficacy and safety of the Chidamide in a case series of 27 patients with first-time diagnosed T-ALL.®), were included in the present study. Patients provided informed consent to receive Chidamide. We excluded patients who received Chidamide for <24 weeks and who abandoned treatment at any point of therapy without achieving any endpoint. The research protocol was approved by the ethics committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University. All data were prospectively collected and analyzed.From July 1, 2016, to May 31, 2021, 235 children were diagnosed with T-ALL in the SCCLG-2016 cooperation group. The SCCLG-2016 ALL protocol  was adopThe diagnosis of T-ALL was based on morphological French-American-British criteria: negative staining for myeloperoxidase or Sudan black and immunophenotypical expression of T- cell surface antigens CD7 or CD2, often with CD5, CD3, CD4, or CD8 in more than 20% of blast cells and nuclear deoxynucleotide transferase or intracytoplasmic CD3 in more than 10% of blast cells. Unequivocal cases were reviewed centrally. Immunophenotyping was performed according to the European Group for the Immunological Characterization of Leukemias or World Health Organization 2008 criteria.9/L and platelets ≥ 100 × 109/L). Minimal residual disease (MRD) in bone marrow aspiration specimens was assessed by multiparameter flow cytometry with a sensitivity of 0.01%. flow cytometry MRD was analyzed according to a previous French multicenter study for pediatric and adult ALL. MRD was analyzed by Kaluza software  or CellQuest software . Reagents were provided by BD Biosciences  and Beckman Coulter Commercial Enterprise (China) Co., Ltd.CR was defined as bone marrow blast cells ≤5%, absence of extramedullary disease and recovery of peripheral blood counts , no partial bone marrow remission (M3 [≥25% blasts] or MRD ≥ 10%) on Day 15, no bone marrow remission  after induction phase 1 (Day 33), and/or positive MLL gene rearrangement, low diploidy, iAMP21 or IKZF1 deletion, MEF2D rearrangement and TCF3-HLF gene fusion, and/or mediastinal tumor lesion evaluated on Day 33 without reduction to 1/3 of the original tumor size or tumor still exists before consolidation therapy. The remaining patients were considered IR.After the seven-day prednisone test and dexamethasone-based four-drug induction , patient2 of Cyclophosphamide (CTX) was added to the traditional BFM induction therapy . Total-body irradiation (TBI) was recommended in peripheral blood stem cell (PBSC) match sibling donor transplantation.Transplantation conditioning regimens for T-ALL were myeloablative, which included: Busulfan 12.8 mg/kg, Fludarabine 120–150 mg/mChidamide was used at a dose of 0.5 mg/kg patient weight every 4 days in combination with chemotherapy for patients with T-ALL undergoing maintenance therapy or started at 4–8 weeks post-HSCT for 2 years.Overall survival was measured as the time from the diagnosis of T-ALL to death or last follow-up. OS was calculated from diagnosis to death (from any cause). Event-free survival (EFS) was measured as the time from achieving CR to relapse, treatment related death or last follow-up. The “event-free” duration was defined as the time from diagnosis to the date of treatment-related death or relapse or until the date of the last contact in CR. For all analyses, patients lost to follow-up were censored at the time of their withdrawal. The survival curves were analyzed using the Kaplan–Meier method.P < 0.05 was considered to indicate statistical significance.Follow-up data were updated as of March 2022. Toxicity was graded based on the modified Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Statistical analysis was performed by using SPSS 23.0 . In total, 27 patients were treated with Chidamide, of whom 12 started post-HSCT and 15 received Chidamide during post-remission maintenance therapy. The median follow-up period was 37.8 months (9.5–67.9 months). 18 patients were not older than 10 years old. Male to Female ratio was 8: 1. Three of them were diagnosed with CNS involvement and stratified as HR at the first diagnosis. According to the risk stratification criteria, 18 patients were treated according to the protocol of HR T-ALL, among which 12 patients received HSCT. The characteristics of individual patients treated with Chidamide are shown in Up to the observation time points, 25 of the children receiving Chidamide were in continuous remission. The survival curves are shown in n = 7), peripheral blood stem cell (n = 4), and mixed graft . The conditioning regimens (n = 2) or MUD transplantation (n = 1) . One was HLA-matched unrelated donors (MUDs), and 8 were mismatched unrelated donors. Two donors were matched sibling donors (MSDs). One donor was haploidentical related donors. The sources of HSCs used were cord blood (regimens  were cla (n = 1) . Five caIn total, 12 patients accepted HSCT at their first complete remission, and accepted Chidamide post-HSCT regularly. The overall survival and event free survival rate were 83.33 and 85.7% at 64.2 months after transplantation , respectn = 6), neutropenia (n = 9), thrombocytopenia (n = 9), renal impairment (n = 1) and infection . The adverse events were all reversible when administration of Chidamide was stopped, and Chidamide therapy was continued with previous doses until relapse, or the end of maintenance therapy, or 2 years post-HSCT.No death was reported over the first year of Chidamide combination therapy and subsequent deaths were all related to disease progression. The adverse events observed during treatment were nausea or vomiting  that the relapse rate in the high-risk group with or without HSCT was reduced and (ii) that a few severe adverse effects were found in the Chidamide group for post-HSCT and post-remission maintenance in pediatric T-ALL.The long-term survival of patients with T-ALL relapse has been lower than 40%, even with intensive chemotherapy or post-HSCT . The sigNOTCH mutation  protocol-based CCLG-ALL2008 protocol in China, relapse cases were reported mostly ultra-early , and early . Only less than 5% of relapse cases occurred after more than 36 months . These p9/L and 4 × 109/L is safe and efficient for maintenance therapy.The dose of Chidamide was 0.5 mg/kg in this study. We adopted this dosage from previous studies, which included children younger than 18 years old . The plaThere are some limitations in this study. Considering the limited number of cases, a larger number of patients should be enrolled to further analyze the efficacy of Chidamide in T- ALL relapse prophylaxis. Furthermore, it was difficult to analyze the efficacy differences between subgroups based on, for example, gene mutation, risk stratification, and early T-cell precursor ALL . To furtIn conclusion, we reported a series of cases of firstly diagnosed T-ALL who received Chidamide during maintenance regimens or post-HSCT with a remission rate of 95% in more than 3 years, showing promising efficacy in relapse prophylaxis. Maintenance regimens containing Chidamide are well tolerated in T-ALL children. The benefit of Chidamide in prolonged EFS deserves further study and confirmation by large-scale prospective clinical trials.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.J-PF designed the study. X-YL and X-WH analyzed the data and wrote the manuscript. X-YL, X-WH, and KH contributed to the retrieval and analysis of essential data. All authors contributed to the charts, critical revision, and final approval of the manuscript.


In vitro, flow cytometry, Western blot, and other experimental methods were performed to evaluate the antitumor effect of miR-563 loaded exosomes. In in vivo, the LC mouse model was used to observe the effect of the prepared exosomes. The safety of using this exosomes was accessed by liver function test, hematological analysis, and H&E staining in major organs of the mice. Our findings indicated that the miR-563 loaded engineered exosomes inhibit the A549 cells growth in vitro, by inhibiting the tumor cell proliferation, migration, and invasion and promoting apoptosis. In in vivo, these engineered exosomes were enriched in tumor tissue after injecting to LC model mice and impacted tumor tissue by inhibiting the tumor volume and tumor weight. Importantly, our study indicated that miR-563 loaded engineered exosomes have the potential for clinical application for LC treatment with acceptable safety profiles. Our findings indicate a novel potential therapeutic target for lung cancer patients by miR-563 loaded engineered exosomes.The malignancy of lung cancer (LC) is serious in the world. Exosomes are well-known natural nanovesicles, which are reported to have the potential to carry functional miRNAs as natural carriers and deliver chemotherapeutic drugs. However, the safety and functions of the engineered exosomes for delivering miRNA for the treatment of LC remain to be evaluated. In this study, we found that miR-563 is related to lung cancer from GeneCard database. RT-qPCR and in situ hybridization (ISH) were used to assess miR-563 expression in clinical samples. We prepared and verified the engineered exosomes loaded with miR-563 both  Lung cancer (LC) seriously threatens human health , 2. The in vivo due to their filling in microcapsules or exosomes [Micro-RNAs (miRNAs) were classical noncoding RNAs with17–24 nt . MiRNAs exosomes . MiRNAs exosomes . And, miexosomes –13. Addiexosomes and repreexosomes .in vivo is longer, greatly increasing the chance of this molecule meeting with target cells [Exosomes (Exo) have unique functions, which can be used as a personalized carrier for treating diseases by targeted drug delivery. They are produced in cells and recognized by the immune system as “self,” which improves their stability in serum and helps to deliver signals to target cells. Furthermore, compared with artificial nanocarriers, their circulation time et cells . The feaet cells . Nie et et cells  preparedin vitro and in vivo to observe its influence on tumors and explore its mechanism. Furthermore, we assessed the safety of the engineered exosome in healthy mice.In order to find an engineered exosome with clinical potential for the treatment of LC, we obtained the miR-563 from GeneCard by searching the keywords “lung cancer” and verified it in clinical samples. Previous studies found that miR-563 can inhibit the growth of LC . In thishttps://www.genecards.org/). We entered the keyword “lung cancer,” and the lung cancer-associated gene symbols were obtained, which contained 1,053 miRNAs. Then, we used the relevance score ≥2.0 as the cutoff value and obtained 330 miRNAs. According to the experimental purposes, and determination by literature search screening and subsequent clinical sample testing, miR-563 with reduced expression in LC was selected as the target miRNA of the present study.We obtained the LC-related genes from GeneCard . RT-PCR and ISH were used to test the miR-563 expression in the clinical samples. The tissues were taken from the patients who underwent LC tissue resection in the Second Hospital of Nanjing. All patients were confirmed by diagnosis histopathology. This study was conducted under the supervision of the Second Hospital of Nanjing after the World Medical Association, and all patients have signed informed consent.2 atmosphere.A549 cells were cultured in RPMI-1640 , and the complete medium contains 10% fetal bovine serum . Then, the cells were put at 37°C and 5% COg for 15 min and 16,000 × g for 20 min). Then, pellets were centrifuged again for 90 min at 100,000 × g at 4°C. EV solutions were filtered through a 0.22 μm syringe filter for future use.EVs were isolated by differential ultracentrifugation from cell culture medium. Firstly, low-speed centrifugation was used for the depletion of cells and cell debris  were mixed with 400 nM of Cy5-labeled miR-563 in 1 mL phosphate buffered saline (PBS). Then, the mixture was electrophoresed at 400 V, 50 sf, three cycles by 30 ms pulse/2 s pause. Then, the exosome samples were diluted and centrifuged at 110,000× g for 70 min.Exosomes  was used to mark the engineered exosomes. A549 cells were coincubated with stained Exo. 24 h later, 4′, 6-diamidino-2-phenylindole (DAPI) was used for marking the nucleus. We observed and recorded the PKH26 in A549 cells in each group by confocal microscope .CCK-8 was used for the proliferation ability of A549 cells after treated with the Exo , and the5 cells were collected and suspended in PBS. Then, Annexin-V and propidium iodide were added and incubated for for 20 min. After that, 480 mL binding buffer was supplemented into cell suspension after a 10-min incubation. At last, the cell apoptosis ratio was analyzed by FACS Calibur Flow Cytometer .In brief, 5 × 10−△△Ct method.We collected total RNA in cells and tissues using Trizol reagent , and the thermoscript RT-PCR synthesis kit  was used to synthesize the first strand cDNA. The ThermoScript RT-qPCR kit was used. The primers used were as follows : miR-563 forward: 5′-AGGTTGACATACGTTTCCC-3′ and reverse: 5′-TCGTATGCCGTCTTCTGCTTGT-3′; U6 snRNA was used as an internal reference in this manuscript: forward: 5′-GACAATCCTAGACTAGCTTACGA-3′ and reverse: 5′-CATGGCACAAGTCATAAGCA-3′. The relative expression analysis of genes was analysis by the 2MiRNA ISH was carried out according to the kit instructions . miR-563 probe: Digoxin-5-GACAATCCTAGACTAGCTTACG-3, Digoxin. U6 probe: Digoxin-5-CACGAATTTGCG TGTCATCCTT-3, and negative control probe: Digoxin-5-AGGTTGACA TACGTTTCCC-3-Digoxin.The exosomes were fixed in 2% paraformaldehyde and 0.1 M phosphate and adsorbed to a 2 Formvar-carbon-coated grid. Exosomes were viewed with a transmission electron microscope using Jeol JEM-1400  operating at 80 kV. The size of exosomes was analyzed using a Zetasizer Nano ZS system .β-actin . The membrane was washed for 3 times and incubated with the corresponding secondary antibody. The protein bands were observed with ECL chemiluminescence kit .The whole extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) in an electrophoresis instrument  and then transferred to a polyvinylidene difluoride (PVDF) membrane. Next, the membrane was washed in TBS Tween 20 and then incubated with primary antibodies against CD63 , TSG101 , Bax , Bcl-2 , cleaved-caspased3 , cleaved-caspase9 , MMP2 , MMP9 , and 5) were incubated to reach 70% confluence. After 24 h treatment with loaded engineered Exo and cultured at 37°C for another 4 h, a scratch wound was created with sterile pipette tip, and the scraped monolayer cell image was recorded.A549 cells  was used to assess the enrich of Exo.BALB/Tumor tissues and other organ samples were collected and fixed in 4% paraformaldehyde. Histological examination was performed in line with a conventional method. H&E and PCNA staining were performed. The slides were scored and identified in a blinded manner.One-step TUNEL Apoptosis Assay Kit from Beyotime Biotechnology  was used for staining. Cells were exposed to the TM red-labeled dUTP after fixed in PBS with 4% paraformaldehyde. And then, samples were counterstained with DAPI. TUNEL positive nuclei were identified and analyzed by fluorescence microscopy.To assessed liver function, mouse serum was used to detect the enzymatic activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). The blood of mice was collected in centrifuge tubes. After centrifugation, the serum was collected and stored at 4 degrees for detection as soon as possible. Serum from mice was assayed using ALT, AST, and ALP activity assay kits . The light absorbance was measured at 510 nm with a Multiskan MK3 .Blood Biochemical Examination includes the red blood cell (RBC), white blood cell (WBC), and platelet (PLT) count. Blood samples of mice were collected, and centrifugation was used to harvested serum samples for test.T-test or one-way analysis of variance was used for comparison between two or more groups. P < 0.05 means statistically significant difference.Data were presented as mean ± standard deviation (SD) from at least 3 independent experiments. GraphPad Prism 5.0 software was used for statistical analysis. In order to find an engineered exosome with clinical potential for the treatment of LC, we obtained the miR-563 from GeneCard by searching the keyword “lung cancer” and then determined by literature search screening. Subsequently, the clinical sample was tested. We analyzed the lung cancer tissues and adjacent tissues by RT-PCR and ISH for the potential effect of miR-563 in LC. miR-563 mRNA expression was downregulated in the cancer tissues by more than 3-fold compared with the control group . SubsequIn order to determine the effect of miR-563 in clinical use, we prepared the engineered Exo loaded with miR-563. Transmission electron microscopy (TEM) was used to analyze the prepared engineering Exo. Under electron microscope, these exosomes appeared as disc-shaped with an average particle size of 30–150 nm . SubsequWe used the fluorescent dye PKH26 to label Exo, and A549 cells were incubated with the PKH26-miR-563 Exo to verify the specific uptake capability of A549 cells. After ingestion, we first observed the quantity of exosomes getting into cells by assessing the fluorescence intensity. We evaluate the A549 cells' uptake capability to miR-563 loaded engineered Exo. It can be seen from the results that the red fluorescence intensity of the ingestion group is the highest . In addiTo explore the role of miR-563 loaded engineered Exo in LC, we first assessed the proliferation and apoptosis of the cancer cells. We investigated the effect of miR-563 loaded engineered Exo on A549 cells proliferation by CCK-8 and EdU analysis. After coincubation with miR-563 Exo for 72 h , which rWe explored whether the miR-563 loaded engineered Exo inhibits the cell migration and invasion of A549 cells. It was found that the wound closure of A549 cells treated with miR-563 Exo was significantly delayed , which iin vivo experimental results suggested that miR-563 loaded engineered Exo promoted the apoptosis of tumor cells and exhibited an antitumor effect in vivo.We continued to evaluate the biodistribution and tumor penetration of miR-563-loaded engineered Exo in mice with tumors. miR-563 loaded engineered Exo labeled with PKH26 was easily found in model mice . In Figuc mice at a dosage as previously described. Compared with PBS and unloaded Exo group, no change in the body weight was observed (The toxicity is another key parameter of an excellent delivery vehicle. To evaluate whether miR-563 loaded engineered Exo was safe, we assessed the systematic toxicity of miR-563 loaded engineered Exo in healthy BALB/observed . We furtobserved , and theobserved . In addiWith the constant development of nanotechnology for the treatment of cancers, nanomaterials have been widely used in various fields. Exosomes are natural macromolecular substance carriers between human cells, which affects cell-cell communication . Exosomein vitro. Furthermore, the tumor inhibitory effect of miR-563 loaded engineered Exo was evaluated in vivo. The lowest tumor volume and weight were observed in mice treated with prepared Exo compared with the PBS and unloaded exosomes, which showed significant antitumor efficiency of miR-563 loaded engineered Exo. In vivo studies also showed the effective delivery of miR-563 to tumor tissues and induced tumor cell apoptosis mediated by miR-563 loaded engineered Exo.We first assess the miR-563 by RT-PCR and ISH in LC tissues and adjacent tissues. We found that miR-563 levels were downregulated in the cancer tissues. Next, we successfully constructed miR-563-loaded engineered Exo to explore whether the miR-563 has the potential for the treatment of LC. Our findings suggested that miR-563 loaded engineered Exo were specifically uptaken by A549 cells. Internalization of the miR-563 loaded engineered Exo in tumor cells influenced the cell behaviours. Our results demonstrated that miR-563 loaded engineered Exo was able to inhibit the tumor cell growth Furthermore, after fine modification, nanodrug carrier can combine with cancer cell membrane, microenvironment, or cytoplasm, so as to deliver high concentration drugs to targeted cancer cells and reduce the toxicity to normal cells –28. In o


In this work, an AlGaN-based Deep-Ultraviolet Light-Emitting Diode structure has been designed and simulated for the zincblende and wurtzite approaches, where the polarization effect is included. DFT analysis was performed to determine the band gap direct-to-indirect cross-point limit, AlN carrier mobility, and activation energies for p-type dopants. The multiple quantum wells analysis describes the emission in the deep-ultraviolet range without exceeding the direct-to-indirect bandgap cross-point limit of around 77% of Al content. Moreover, the quantum-confined Stark effect on wavefunctions overlapping has been studied, where Al-graded quantum wells reduce it. Both zincblende and wurtzite have improved electrical and optical characteristics by including a thin AlGaN with low Al content. Mg and Be acceptor activation energies have been calculated at 260 meV and 380 meV for Be and Mg acceptor energy, respectively. The device series resistance has been decreased by using Be instead of Mg as the p-type dopant from 3 kΩ to 0.7 kΩ. Nowadays, III-nitride is one of the most important semiconductor families for device development, such as transistors, lasers, photodetectors, and light-emitting diodes (LEDs) . DiffereIt is remarkable to mention that, despite external quantum efficiency (EQE) remaining under 10% ,16,17, sFurthermore, recently cubic phase has demonstrated the capability to achieve high hole concentration due to its lower activation energy ~100 meV for p-dopant Mg in zb-GaN.  If the h0.7Ga0.3N with p-type substituting dopant (Be or Mg) considered for simulation to obtain the activation energy for the dopants.It is critical to focus on the p-doping performance of zb-AlGaN since it is essential to achieving zb-AlGaN-based DUV-LED and other optoelectronic devices. The substituting Be and Mg defect formation energy as a function of the Fermi level calculated under N-rich conditions are shown in 0.2Ga0.8N to 370 meV in zb-Al0.8Ga0.2N, which is lower /(32π2ε2ħ2), where m* is the effective mass, e is the elementary charge, and ε is the dielectric constant. Since GaN has a larger dielectric constant but similar effective mass for holes (compared to AlN), the activation energy of Mg and Be in GaN is smaller and would increase when more Al content is included.This behavior shows that Mg remains a shallow acceptor in zb-AlGaN. As a main group-II element, Be is also expected to act as an effective p-type dopant by substituting Al or Ga, though no experiment has proven it until now. Compared to Mg, Be in zb-AlGaN shows higher formation energy but lower activation energy which ranges from 75 meV in zb-Alous work  and demoous work  (m*e4)/(0.70Ga0.30N presents a direct bandgap while zb-Al0.85Ga0.15N exhibits an indirect bandgap due to the conduction band minimum (CBM) shift from Γ point to X point. Note that the actual band gap values are underestimated for zb-AlxGa1-xN, which corresponds to a well-known DFT limitation. Nonetheless, the band features, such as the direct/indirect gap predicted here, are pretty accurate. Further data interpolation (0.77Ga0.23N), exhibiting a maximum Al content around 0.77, which is valuable guidance to design zb-AlxGa1-xN based DUV range LED and other optoelectronic devices.In the context of DUV-LED application, the direct band gap of the active materials should be ~4.8 eV. The zb-GaN has a direct bandgap of 3.2 eV, and zb-AlN shows an indirect bandgap of 5.0–5.3 or even 6.0 eV considering the conduction band edge at Γ-valley for indirect-gap alloys . Thus, ipolation b revealsP is the parameter to determine, x is the Al content in the alloy, and b is the bowing factor. Excluding the Energy gap (Eg), all bowings factors were not considered, assuming linear dependency on the Al molar fraction. Moreover, the electron affinity has been calculated using the bandgap offset of 0.75 from ΔEc/(ΔEc + ΔEv) [Besides the direct-to-indirect cross-point limit, the AlN effective masses and carrier mobilities were calculated by DFT. Binary AlN and GaN values were used to establish a cubic III-nitrides-based alloy simulation set. The zb-LED structure (c + ΔEv) . The zb-r is the material refractive index, c and fv are the fermi functions in conduction and valence band, m0 is the electron mass in kg, ε0 is the vacuum permittivity, and rm is reduced effective mass obtained from Equation (3).cm and vm are effective masses in the conduction and valence band, respectively, this reduced effective mass is used to calculate ρr, the density of states given by Equation (4).Capture-Scape rates are simulated to present the influence on the active region of the polarization effect. For SILVACO simulation, the Kronig–Penney model for drift-diffusion, Fermi–Dirac for carrier statistics considering incomplete ionization, and the two-band zb model for gain and radiative recombination have been chosen. For zb and wz-approaches, the radiative recombination dependent on polarization  and spontaneous emission rate are modeled using Equation (2).The scaling factor of polarization is set to zero for the zb approach and 1.0 for wz to add the polarization effect. Moreover, for the strain models for zb and wz, strain tensor calculations are performed in the SILVACO environment by selecting the strained two- and three-band models  for gain and radiative recombination. Before SILVACO simulation, a finite square well analysis based on effective mass approximation was performed. The first energy level in the quantum well (QW) is calculated by solving the Schrödinger Equation, ensuring wavefunction continuity across the entire quantum well/quantum barrier (QW/QB) heterostructure. Energy is calculated using the Newton–Raphson numerical method to solve Equation (5) .(5)tanθThis quick analysis has been performed to determine a big picture of the thickness and the Al molar fraction in the QW necessary to emit in the DUV range.55Ga0.42N/Al0.75Ga0.25N multiple quantum well (MQW) with three QWs to emit around 275 nm with the zb-AlGaN approach. A thin contact layer (P1) has been implemented to mitigate the low light extraction efficiency due to the high transverse-magnetic polarized light dominance in high Al-rich AlGaN-based MQW structures [0.50Ga0.50N with p = 2 × 1019 cm−3. The electron blocking layer (EBL) was considered with p = 2 × 1019 cm−3 [18 cm−3 [0.70Ga0.50N with electron concentration n = 2 × 1019 cm−3 to broaden the electric field along the active region.The structure to simulate using SILVACO Atlas  was deteructures . The hol019 cm−3 , lower t019 cm−3 ,38, and [18 cm−3  were als, it can be seen that there is a remarkable reduction in activation energy in the p-layer by doping with Be instead of Mg. DFT calculations for zb Mg doping simulation presented an activation energy of 340 meV for p-Al0.70Ga0.30N, whereas the Be doping shows a lower activation energy of around 200 meV. As shown in s-d and p-d orbital interaction [d-orbital in Al, so AlN shows an indirect bandgap, similar to Silicon. In GaN, due to the occupied d-orbital in Ga, the s-d and p-d orbital couplings push the conduction band energy at X valley up but leave the Γ valley intact. The calculated direct bandgap cross-point limit provides essential material information for AlGaN-based device design. From the data in Figure 1In addition to band gap directivity and p-doping capability, carrier mobility is another figure of merit for the AlGaN LED device application. L is the thickness to integrate, and the min function denotes de overlapping for electron and hole wavefunctions. As the QW thickness increases, the QW emission wavelength also increases due to the thickness dependence of the quantum-confined Stark effect (QCSE). As it is well known, if an electric field is induced in a confined region such as QW, this QCSE provokes a shift in the wavefunctions (electron to the left and holes to de right), reducing the overlapping. This effect has been compensated by grading Al content in each QW (wz- structure with graded QWs) from 0.55 to 0.60 Al molar fraction with a thickness of 1.5 nm [The finite square well calculations are shown in f 1.5 nm .As shown in Additionally, to enhance the electrical characteristics of the DUV-LED, the content of Al in the P1 layer is varied and compared to the structure without the P1 layer and a p-GaN thin contact layer. I-V curves for these simulations are presented in −19 cm−3 is a technological challenge in Al-rich layers, it is important to mention that epitaxial growth over the p = 2 × 1019 cm−3 has been explored experimentally for the zb-GaN [By selecting Be instead of Mg as a p-type dopant, the Rs of the structure is reduced approximately three times. e zb-GaN  and Mg sThe simulations show that zb-AlGaN is a candidate for the development of DUV-LED. As presented in this work, a simple approximation, such as SQW, shows that the range of luminescence lies under 275 nm if the QW thickness stands under 1.5 nm and the Al content is limited by the direct-to-indirect cross-point limit of around 77%. Moreover, highly-doped cubic III-nitrides are possible due to their lower activation energy for dopants. The activation energy presented more than 100 meV reduction from 260 to 370 meV using Be instead of Mg. The presented electrical and optical characteristics have been improved for the zb-AlGaN-based LED structure by modifying the p-region modification using a thin AlGaN-based contact layer, where Be, as the p-type dopant, exhibits an LED performance enhancement. A reduction of the Rs from 2.35 kΩ to 0.72 kΩ without the polarization effect in the structure has been determined by considering Be-dopant instead of Mg, presenting Be as an alternative to improve DUV-LED performance by modifying the p-type layers. Moreover, by grading the QW, the polarization effect on bands has been mitigated, enabling future research.